Water-soluble photoluminescent D -mannose and L -alanine functionalized silicon nanocrystals and their application to cancer cell imaging † | 8427 Journal

Herein, we report the straightforward synthesis, photoluminescent properties, and cell imaging studies of D -mannose and L -alanine functionalized silicon nanocrystals (SiNCs). Tailoring nanocrystal surface functionalization is essential to interfacing SiNCs with their environment and rendering them stable – surface modi ﬁ cation also o ﬀ ers the opportunity to target speci ﬁ c cell types for imaging. A simple and versatile surface modi ﬁ cation procedure was developed to tether biomolecules onto the SiNC surfaces and render them water-soluble. The presented approach is precious metal-catalyst free, straightforward, and provides carbohydrate and amino acid functionalized SiNCs. The functionalized SiNCs have been investigated by ﬂ uorescence microscopy and our results indicate that they can be internalized by MCF-7 human breast cancer cells as shown in the cell imaging studies. The obtained SiNCs were characterized using FTIR, XPS, PL, and TEM.


Introduction
Luminescent quantum dots (QDs) have been proposed as additional type of materials to uorescent organic dyes for bioimaging applications. [1][2][3][4][5] Photobleaching is among the most important challenges associated with organic dyes and limits long-term in vitro and in vivo bioimaging. 6 QDs do not photobleach, exhibit tunable optical properties, 1 and possess tailorable surface chemistry. 7 However, II-VI QDs (e.g., CdSe/ZnS QDs) are cytotoxic and hence are of limited practical utility for applications involving biological systems. [8][9][10][11] Silicon nanocrystals (SiNCs) have attracted considerable interest in recent years for biological applications [12][13][14][15][16] because of their established biocompatibility, 17 abundance, and tunability. 18 Because oxidation can change and even quench SiNC photoluminescence (PL), it is crucial that their surfaces be passivated. Unfortunately, protocols typically used to modify II-VI QDs are not readily compatible with SiNCs and alternative methods must be devised. To date, most approaches used to modify SiNC surfaces have aimed to introduce long chain alkyl groups rendering NCs hydrophobic and soluble in common organic solvents. Materials modied in this way are typically insoluble in water and their compatibility with biological systems is limited unless further modication is performed (e.g., polymer coating). 19 Drawing on established approaches for rendering other QDs water-soluble, polar functionalities (e.g., -NH 2 and -COOH) have been tethered to SiNC surfaces. 7,[20][21][22] Unfortunately, amine terminated SiNCs are cytotoxic 23 and carboxylic acid terminated particles can be challenging to prepare and oen show weak PL. 24 In this context, it is useful to explore alternative surface functionalities. Among potential candidates, carbohydrates and amino acids are particularly attractive because these moieties are water-soluble, can be recognized and taken up by cells, and they do not compromise SiNC optical response. While reports describing carbohydrate functionalized SiNCs have appeared, 25,26 these methods typically involve laborious multi-step procedures, as well as costly and potentially toxic noble metal catalysts 27 that can compromise SiNC optical response. 28 To our knowledge, there are no reports of amino acid terminated SiNCs. In this study, we report a straightforward synthetic protocol for preparing water-soluble SiNCs functionalized with D-mannose and L-alanine and demonstrate a prototype application of these particles as luminescent imaging agents for the visualization of MCF-7 human breast cancer cells.

Synthesis of hydride-terminated SiNCs
3.0 g of HSQ was transferred to a quartz boat and thermally processed under slightly reducing conditions (i.e., owing 5% H 2 /95% Ar) at 1100 C for 1 h in a tube furnace. Aer the resulting product was cooled to room temperature the amber solid consisting of oxide-embedded SiNCs was ground using an agate mortar and pestle. To obtain freestanding hydrideterminated SiNCs, the ground SiNC/SiO 2 composite (0.30 g) was etched using 9 mL of a 1 : 1 : 1/v : v : v mixture of H 2 O : ethanol : 49% HF solution. The mixture was stirred for an hour under ambient conditions to remove the SiO 2 matrix. The resulting hydride terminated SiNCs were extracted using three 10 mL portions of toluene. The product was centrifuged at 3000 rpm for 10 min and the supernatant was discarded.

Synthesis of chloride-terminated SiNCs
PCl 5 (0.30 g, 1.4 mmol) was added to a dry Schlenk ask containing hydride-terminated SiNCs (ca. 60 mg) dispersed in toluene (10 mL) to form a cloudy orange suspension. The resulting mixture was stirred for 1.5 h under argon atmosphere. Following the reaction the product mixture appeared as a light yellow clear solution. The solvent and reaction byproducts were then removed in vacuo to yield chloride terminated SiNCs (Cl-SiNCs) as an orange solid.
Synthesis of D-mannose functionalized SiNCs 1 (0.10 g, 0.4 mmol) was transferred to a Schlenk ask containing Cl-SiNCs (vide supra). 12 mL of anhydrous toluene were added to the reaction ask. The resulting mixture was stirred for 16 h at 40 C under inert Ar atmosphere. Subsequently, the toluene was removed from the crude product mixture under vacuum and the resulting particles were dispersed in 10 mL methanol. Solid sodium methoxide was added to the methanol solution until the pH $ 9. The resulting solution was stirred in air at ambient temperature for 12 h and then neutralized using Amberlite IR-120 ion exchange resin (acid form). The neutralization was monitored using pH paper. The resulting neutral solution was concentrated to yield a solid using a rotary evaporator and then functionalized SiNCs were redispersed in water and the exchange resin was removed via gravity ltration.

Synthesis of L-alanine functionalized SiNCs
L-alanine methyl ester (0.08 g, 0.6 mmol) was added to a dry Schlenk ask containing Cl-SiNCs (vide supra). 12 mL dry toluene were added to the reaction ask and the resulting mixture was stirred for 16 h at 40 C under inert Ar atmosphere. Following the reaction the solvent was removed using a rotary evaporator. The resulting solid residue was redispersed in 5 mL of methanol. 2.5 mL of 1 M aqueous NaOH were added to the alanine methyl ester functionalized SiNCs. The solution was then heated to reux and stirred for 1 hour followed by cooling to room temperature. The resulting solution was neutralized using Amberlite IR-120 ion exchange resin (acid form), and the neutralization process was monitored using pH paper. The solution was concentrated to yield a solid using rotary evaporator. The SiNCs were redispersed in water and the exchange resin was removed via gravity ltration.

Synthesis of pentanoic acid functionalized SiNCs
Immediately aer etching 0.2 g of SiNC/SiO 2 composite, the resulting hydride-terminated SiNCs were isolated and redispersed in 15 mL dry toluene in a Schlenk ask equipped with a stir bar. Azobisisobutyronitrile (AIBN, 10 mg) and 4-pentenoic acid (4 mL) were added to the ask and the mixture degassed by performing three freeze/pump/thaw cycles using an Ar charged Schlenk line. The solution was stirred at 65 C for 15 h aer which time the reaction mixture was transferred into test tubes and centrifuged at 3000 rpm. The supernatant was discarded and the solid product was puried by three successive dispersion/precipitation cycles by the addition of ethanol/pentane mixture as the solvent/antisolvent and centrifugation.
Cell studies 10 mL of the solution of mannose functionalized SiNCs (620 mg mL À1 ) were added to 300 mL medium containing MCF-7 cells (6000 cells per well). The same concentration of solutions of alanine and pentanoic acid functionalized SiNCs were prepared and added to MCF-7 cells respectively. The cells were incubated at 37 C for 24 hours. Subsequently, the medium was removed upon washing with phosphate buffered saline (PBS). Cells were "xed" upon incubation with paraformaldehyde for 30 minutes. Fixed cells were washed with PBS three times and a small drop of anti-fade solution was added to each slide. A coverslip was placed over the specimen and the edges were sealed with clear nail polish.

Characterization
Fourier Transformation Infrared Spectroscopy (FT-IR) was performed using a Nicolet Magna 750 IR spectrometer. Photoluminescence (PL) spectra of solution samples were acquired using a Varian Cary Eclipse Fluorescence Spectrometer. High resolution (HR) TEM images were obtained from Hitachi-9500 electron microscope with an accelerating voltage of 300 kV. TEM samples were prepared by dropcoating freestanding SiNC suspensions onto a holey carbon coated copper grid and the solvent was removed under vacuum. The NC size was averaged for 200 particles using Image J soware (version 1.45). The HRTEM images were processed using Gatan Digital Micrograph soware (Version 2.02.800.0). X-ray photoelectron spectroscopy (XPS) analyses were performed using a Kratos Axis Ultra instrument operating in energy spectrum mode at 210 W. Samples were prepared as lms dropcast from solution onto a copper foil substrate. The base and operating chamber pressure were maintained at 10 À7 Pa. A monochromatic Al K a source (l ¼ 8.34Å) was used to irradiate the samples, and the spectra were obtained with an electron take-off angle of 90 . To minimize sample charging, the charge neutralizer lament was used when required. Survey spectra were collected using an elliptical spot with major and minor axis lengths of 2 and 1 mm, respectively, and 160 eV pass energy with a step of 0.33 eV. CasaXPS soware (VAMAS) was used to interpret high-resolution (HR) spectra. All spectra were internally calibrated to the C 1s emission (284.8 eV). Aer calibration, a Shirley-type background was applied to remove most of the extrinsic loss structure. The FWHM for all the tted peaks was maintained below 1.2 eV.
Relative photoluminescent quantum yields were determined using methods adapted from the work of Williams et al. 31 Cyclohexane solutions of 9,10-biphenylanthracene and absolute ethanol solutions of coumarin 1 were used as uorescent standards. Functionalized Si-NCs were dissolved in methanol. All organic solvents were passed through a column of anhydrous magnesium sulfate to remove residual moisture immediately prior to solution preparation. Stock solutions were prepared by dissolving 10 mg of the organic dye in the appropriate solvent. Solutions were subsequently ltered through a membrane lter to remove suspended impurities. A series of solutions with absorbances within the range of 0.01 to 0.1 were prepared. PL spectra were acquired for the identical solutions upon excitation at 300 nm. The slopes of plots of the integrated uorescence intensity vs. UV-Vis absorbance intensity curves determined and compared to conrm the quality of the reference curves. In all cases, the experimentally determined quantum yields were in good agreement with literature values: 9,10-biphenylanthracene, 0.9 and coumarin 1, 0.73. The PL spectra of Si-NC solutions and reference samples were acquired under identical conditions. The quantum yields were determined using the following equation: where '4' is the quantum yield, 'm' is the slope of the integrated PL versus absorbance curve and 'h' is the refractive index of the solvent. The subscript 'st' refers to the standard organic dye while 'x' indicates the unknown species to be calculated. Dynamic Light Scattering (DLS) measurements were performed using a Malvern Zetasizer Nano S equipped with a 633 nm laser. All samples were ltered using a 0.45 mm PTFE syringe lter and equilibrated to 25 C prior to data acquisition. A refractive index of 1.460 used for all measurements.

SiNCs synthesis and surface functionalization
Our group has previously reported the preparation of oxideembedded and freestanding SiNCs from the reductive thermal processing of HSQ. 24 This procedure affords well-dened, comprehensively characterized materials with dimensions that depend upon the processing conditions. 32,33 Chloride terminated SiNCs (Cl-SiNCs) are obtained upon reaction of hydride terminated SiNCs with phosphorus pentachloride (PCl 5 ). 34,35 Reaction byproducts and toluene are readily removed from the Cl-SiNCs in vacuo. However, isolation and direct characterization of these particles was impractical given their reactivity. It is these Cl-terminated NCs that were used as the functionalization platform for the present study. Further derivatization of Cl-SiNCs (Scheme 1) was achieved upon direct addition of acetate-protected D-mannose or L-alanine methyl ester hydrochloride followed by neutralization and appropriate deprotection. The resulting functionalized SiNCs were dispersed in water.

Nanocrystal characterization
FTIR spectroscopy provides valuable insight into the nature of the SiNC surface chemistry ( Fig. 1 and S6 †). As expected, the FTIR spectrum of hydride terminated SiNCs (not shown) reveals a characteristic Si-H stretching feature at 2100 cm À1 . 32 The reactivity of Cl-SiNCs precluded isolation and direct characterization; however, comprehensive characterization (vide infra) of the products isolated from subsequent derivatization procedures conrm the NC integrity was not compromised by exposure to PCl 5 .
Functionalization with an acetate-protected D-mannosederived glycoside, L-alanine methyl ester, and pentanoic acid yielded SiNCs displaying FTIR spectra with features characteristic of the target surface groups (see Fig. 1 and S6 †). Among the Scheme 1 Chlorination of hydride-terminated SiNCs and the synthesis of mannose and alanine functionalized SiNCs.
most dominant features in both spectra is an absorption at ca. 1735 cm À1 that is readily attributed to C]O stretching arising from the ester functionalities. Following deprotection of the Dmannose SiNCs the C]O feature was replaced by a broad absorption centred at 3350 cm À1 consistent with the presence of -OH moieties. A comparison of the spectra obtained before and aer deprotection of the alanine methyl ester functionalized SiNCs revealed similar changes.
Among the attractive properties of the present SiNCs that can be applied to biological imaging is their PL response. SiNC PL can be inuenced by surface chemistry although the exact origin of the luminescence remains the subject of much investigation. [34][35][36][37][38] Aqueous solutions of the presented mannose and alanine functionalized SiNCs show blue PL with maximum ca. 415 nm and 400 nm upon excitation at 350 nm (Fig. 2). The PL spectrum of an aqueous solution of pentanoic acid functionalized SiNCs shows a maximum at ca. 640 nm upon excitation at 350 nm. Photoluminescent quantum yields of mannose and alanine functionalized SiNCs have been investigated. The quantum yield of mannose functionalized SiNCs is 13% and of alanine functionalized SiNCs is 17%.
XPS provides information regarding the elemental composition of the functionalized SiNCs, insight into the degree of surface oxidation, and conrmation that the SiNC core remains intact. Survey XP spectra of the presented NCs (Fig. S5 †) showed expected elemental emission features. The emissions at 102.2 eV (Fig. 3A), 102.1 eV (Fig. 3B), and 100.3 eV (Fig. 3C) can be attributed to Si-O, Si-N and Si-C functionalities, respectively. 35 The emission feature at lower binding energy (i.e., ca. 99.3 eV) conrms the presence of a Si (0) core in each sample. The N 1s peak (Fig. 3D) is consistent with the presence of alanine on the particle surface.

Cell study
All three surface modications chosen for the present study rendered SiNCs water-soluble and compatible with cell media. Previously D-mannose surface functionalities proved effective in the targeted imaging of MCF-7 breast cancer cells; 25 alanine and pentanoic acid were chosen for their structural simplicity. MCF-   7 human breast cancer cells are an epithelial human cell line that grows quickly and easily and offers a convenient test imaging system. The uptake of mannose (Fig. 6B) and alanine (Fig. 6D) functionalized SiNCs by MCF-7 breast cancer cells was observed. To investigate if particle uptake resulted from the mannose or alanine functionalization, and not straightforward water solubility, cells were exposed to water-soluble red photoluminescent pentanoic acid functionalized SiNCs (see Fig. S6 and S7 † for detailed characterization). No uptake of pentanoic acid functionalized SiNCs by the MCF-7 breast cancer cell was observed. These observations support the conclusion that mannose and alanine functionalization promoted cell uptake.

Conclusions
In this work, we have demonstrated the synthesis and characterization of water-soluble, photoluminescent functionalized SiNCs bearing D-mannose, L-alanine, or pentanoic acid. These particles were subsequently visualized in MCF-7 human breast cancer cells. Mannose and alanine functionalized SiNCs were found to be taken up by MCF-7 cells allowing imaging; however, the pentanoic acid terminated SiNC did not. This observation indicates SiNCs can be effective luminescent imaging agents and that appropriate surface chemistry is crucial to their effectiveness in this application.